Synthesis and kinetic evaluation of tyrosine analogs---implications for the catalytic mechanism of tyrosine phenol-lyase from Citrobacter freundii

The tyrosine analogs, 2-aza-D,L-tyrosine and 3-aza-D,L-tyrosine, were synthesized, and their steady state kinetic parameters were determined for tyrosine phenol-lyase from Citrobacter freundii. Neither compound was found to be a substrate upon examination using the LDH/NADH-coupled assay. Both analogs were found to be inhibitors of TPL, with 3-azatyrosine exhibiting a K_i of 3.36 mM, while 2-azatyrosine had a pH-independent K_i of 17 μM.; The synthetic usefulness of tyrosine phenol-lyase from Citrobacter freundii was also examined. The amino acids, 2-aza-L-tyrosine and 3-aza-L-tyrosine, were synthesized and their pre-steady-state kinetic parameters were determined. Stopped-flow analysis revealed that both azatyrosines bind to TPL and undergo deprotonation to form quinonoid intermediates with absorbance maxima at 504 nm. At high amino acid concentrations, 3-azatyrosine seems to undergo either elimination or transamination as indicated by a decrease in the absorbance at 504 nm.; The tyrosine analogs, erythro- and threo-β-hydroxy-D,L-tyrosines, were synthesized and their steady state and pre-steady-state kinetic parameters were determined for tyrosine phenol-lyase from Citrobacter freundii . Examination of the hydroxytyrosines, using the LDH/NADH-coupled assay, revealed that neither compound underwent elimination in the presence of TPL, possibly due to steric effects. However, both displayed an inhibitory effect, with K_i values of 0.24 mM and 0.25 mM for erythro - and threo-β-hydroxytyrosines, respectively. Analysis using stopped-flow kinetic methods showed that both compounds were capable of quinonoid intermediate formation, although the formation was quite slow.; Extensive efforts were made to synthesize 1,3,4,5-tetrahydro-2-oxo-L-histidine for evaluation as an inhibitor and mechanistic probe for tyrosine phenol-lyase from Citrobacter freundii. The specific details of the synthetic endeavor are presented, although the total synthesis was never achieved.